Molecular characterisation of the causal agent of bacterial leaf streak of maize NJJ Niemann 21114900 Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae in Environmental Sciences at the Potchefstroom Campus of the North-West University Supervisor: Prof CC Bezuidenhout Co-supervisor: Prof BC Flett May 2015 Declaration I declare that this dissertation submitted for the degree of Master of Science in Environmental Sciences at the North-West University, Potchefstroom Campus, has not been previously submitted by me for a degree at this or any other university, that it is my own work in design and execution, and that all material contained herein has been duly acknowledged. __________________________ __________________ NJJ Niemann Date ii Acknowledgements Thank you God for giving me the strength and will to complete this dissertation. I would like to thank the following people: My father, mother and brother for all their contributions and encouragement. My family and friends for their constant words of motivation. My supervisors for their support and providing me with the platform to work independently. Stefan Barnard for his input and patience with the construction of maps. Dr Gupta for his technical assistance. Thanks to the following organisations: The Maize Trust, the ARC and the NRF for their financial support of this research. iii Abstract All members of the genus Xanthomonas are considered to be plant pathogenic, with specific pathovars infecting several high value agricultural crops. One of these pathovars, X. campestris pv. zeae (as this is only a proposed name it will further on be referred to as Xanthomonas BLSD) the causal agent of bacterial leaf steak of maize, has established itself as a widespread significant maize pathogen within South Africa. Insufficient information about the present distribution of the pathogen is available. The main aim of the study was thus to isolate and characterise the pathogen using molecular methods. Results demonstrated that the causal agent of bacterial leaf streak disease (Xanthomonas BLSD: potentially X. campestris pv. zeae) was widely distributed within the major maize cultivation regions of South Africa. Most of the isolates collected originated from the Highveld maize production provinces (North West, Free State, Gauteng and Mpumalanga provinces) as well as from irrigated maize fields in the Northern Cape province. The XgumD gene marker was used to determine if the isolates belonged to the genus Xanthomonas. The gumD gene fragment is located within the gumB-gumM region of the operon and is conserved among Xanthomonas species. This gene fragment is partially responsible for xanthan production. This marker was amplified from all isolates and a selected number were sequenced. The marker was only able to confirm that the causal agent was a member of the genus Xanthomonas. PCR methods were used for the characterisation of the isolates. This included PCR and sequencing of ribosomal RNA- gyraseB and gumD genes. A fingerprinting method BOX- PCR was also employed. Good quality DNA of sufficient quantities was obtained from the various isolates. Amplification produced no non-specific amplification products. This resulted in good quality sequences that could be analysed using bioinformatics tools. Phylogenetic analyses of the ribosomal RNA and gyraseB genes could not detect differences amongst the 47 Xanthomonas BLSD isolates. However, these genes were able to distinguish between the type strain of these isolates and various Xanthomonas species and pathovars. From all three neighbour joining trees the Xanthomonas BLSD isolates had close association with X. axonopodis pv. vasculorum strain ATCC 35938. For the 16S rRNA gene there exists no sequence differences between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. A single nucleotide difference was observed between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938 for the 23S rRNA gene. The gyraseB gene detected a total of six nucleotide variations between these two Xanthomonas species. For all of the phylogenetic trees there was no clustering of Xanthomonas BLSD with X. campestris pathovars. iv Genetic profiling (via BOX-PCR) based on present/absent analysis revealed no variations amongst the Xanthomonas BLSD isolates. All isolates shared an identical pattern produced by 12 distinct PCR products. This profiling technique did differentiate between the isolates of Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. Their profiles shared common bands, but differed in the number and overall pattern of the bands. These results suggest two main conclusions: (i) Xanthomonas BLSD has a clonal origin with geographical separation not impacting genetic variation. The fact that all the isolates appear to be clonal may imply that when resistant maize cultivars are developed these should be resistant to all isolates of the pathovar irrespective of their geographical origin. This is a suggestion that will have to be corroborated using more isolates and additional genetic fingerprinting techniques (ii) the Xanthomonas BLSD isolates from this study may not belong to X. campestris. Further studies using other markers should be conducted to determine the real identity of Xanthomonas BLSD. Keywords – Xanthomonas, bacterial leaf streak disease, X. campestris pv. zeae; maize, ribosomal RNA; gyraseB; X. axonopodis pv. vasculorum strain ATCC 35938; BOX-PCR profiling; clonal origin. v Table of Contents Declaration……………………………………………………………………………………………………………………………………….ii Aknowledgements…………………………………………………………………………………………….…………………………...iii Abstract………………………………………………………………………………………………………………………..………………….iv List of Tables ……………………………………………………………………………………………………………………....……….viii List of Figures................................................................................................................................ viii Chapter 1 - Literature Review and Research Rationale .............................................................. 1 1.1 The origin of Zea mays subsp. mays .................................................................................. 1 1.2 Maize requirements .............................................................................................................. 2 1.3 Maize production .................................................................................................................. 3 1.4 The South African climate .................................................................................................... 6 1.5 Pathogenicity of Phytobacteria ............................................................................................ 9 1.6 Phytopathogens of maize .................................................................................................. 14 1.7 The Xanthomonads ............................................................................................................ 15 1.8 Xanthomonas BLSD the causal agent of bacterial leaf streak (BLS) of maize ............. 19 1.9 The function of xanthan ..................................................................................................... 20 1.10 Genotypic methods .......................................................................................................... 22 1.10.1 Specific markers ................................................................................................... 24 1.10.2 Ribosomal DNA analyses .................................................................................... 24 1.10.3 Protein encoding genes – GyraseB .................................................................... 30 1.10.4 Genomic profiling - the repetitive extragenic palindromic PCR technique ...... 31 1.11 Research Rationale .......................................................................................................... 32 Chapter 2 - Materials and Methods ............................................................................................. 34 2.1 Sample site and collection ................................................................................................. 34 2.2 Bacterial isolation, purification and pathogenicity testing................................................ 35 2.3 Genomic DNA isolation ...................................................................................................... 36 2.4 DNA amplification ............................................................................................................... 36 2.4.1 XgumD PCR ............................................................................................................ 37 2.4.2 16S rDNA PCR ....................................................................................................... 37 2.4.3 23S rDNA PCR ....................................................................................................... 38 2.4.4 GyraseB PCR.......................................................................................................... 38 2.4.5 BOX PCR ................................................................................................................ 38 2.5 Agarose gel electrophoreses of PCR products ................................................................ 38 2.6 Clean-up of PCR products and sequencing ..................................................................... 39 vi 2.7 Statistical analysis .............................................................................................................